Efficacy of first‐line treatment options beyond RET‐TKIs in advanced RET‐rearranged non‐small cell lung cancer: A multi‐center real‐world study

Abstract Background Although RET‐tyrosine kinase inhibitors (RET‐TKIs) are the preferred first‐line therapy for advanced RET‐arranged NSCLC, most patients cannot afford them. In this population, bevacizumab, immunotherapy, and chemotherapy are the most commonly used regimens. However, the optimal scheme beyond RET‐TKIs has not been defined in the first‐line setting. Methods This retrospective study included 86 stage IV NSCLC patients harboring RET rearrangement from six cancer centers between May 2017 and October 2022. RET‐TKIs, chemotherapy, or one of the combination therapies (including immune checkpoint inhibitor (ICI) combined with chemotherapy (I + C), bevacizumab combined with chemotherapy (B + C), ICI and bevacizumab combined with chemotherapy (I + B + C)), were used as the first‐line therapeutics. The clinical outcomes and safety were evaluated. Results Fourteen of the 86 patients received RET‐TKIs, 57 received combination therapies, and 15 received chemotherapy alone. Their medium PFS (mPFS) were 16.92 months (95% CI: 5.9–27.9 months), 8.7 months (95% CI: 6.5–11.0 months), and 5.55 months (95% CI: 2.4–8.7 months) respectively. Among all the combination schemes, B + C (p = 0.007) or I + B + C (p = 0.025) gave beneficial PFS compared with chemotherapy, while I + C treatment (p = 0.169) generated comparable PFS with chemotherapy. In addition, I + B + C treatment had a numerically longer mPFS (12.21 months) compared with B + C (8.74 months) or I + C (7.89 months) schemes. In terms of safety, I + B + C treatment led to the highest frequency of hematological toxicity (50%) and vomiting (75%), but no ≥G3 adverse effect was observed. Conclusions I + B + C might be a preferred option beyond RET‐TKIs in the first‐line therapy of RET‐arranged NSCLC. Combination with Bevacizumab rather than with ICIs offered favorable survival compared with chemotherapy alone.


| INTRODUCTION
Lung cancer is the leading cause of cancer-related death worldwide, in which non-small cell lung cancer (NSCLC) accounts for 80%-85%. 1,2It is reported that more than half of the NSCLC patients are initially diagnosed at advanced stages, with a 5-year survival rate of 15%-19%. 3Over the last three decades, the identification of oncogene mutations and the development of specific targeted therapies have greatly changed the treatment landscape of advanced NSCLC.For example, the application of EGFR tyrosine kinase inhibitors (EGFR-TKIs) has extended overall survival (OS) by over 2 years, from an average of 10-12 months to 38.6 months. 4,5Subsequently, a series of small-molecule TKIs targeting specific oncogenic drivers, including ALK, ROS1, KRAS, BRAF, MET, and NTRK, were developed and approved as the preferred treatments for advanced NSCLC. 6These progresses have ushered in a "TKI first" era in NSCLC treatment.
The receptor tyrosine kinase rearranged during transfection (RET) gene is a proto-oncogene encoding a transmembrane tyrosine kinase receptor that is crucial for normal embryonic development. 7RET alterations, including mutations, rearrangements, and amplifications, result in ligand-independent constitutive activation of intracellular tyrosine kinases and downstream signaling pathway transmission, which subsequently promote tumor cell proliferation and invasion. 8,9RET gene fusions occur in 1%-2% of NSCLC patients 10,11 and are usually mutually exclusive with EGFR, KRAS, ALK, or ROS1 alterations.RET gene fusions are common in females, never-smokers, and patients with lung adenocarcinomas.RET rearrangements are also associated with a high risk of brain metastases.It has been reported that 46% of NSCLC patients with RET rearrangements develop brain metastases during their lifetime. 12The most common fusion partners of RET rearrangements in NSCLC were KIF5B (71.2%) and CCDC6 (16.85%). 13he treatment of RET-rearranged NSCLC has been evolved from chemotherapy-based combination therapy to multi-kinase inhibitor (MKI) to selective RETtyrosine kinase inhibitors (RET-TKI) in the past decades.Chemotherapy alone or combined with Bevacizumab are standard strategies before the appearance of targeted therapy. 14Platinum-based chemotherapy generated an overall response rate (ORR) of approximately 50% and a medium progression-free survival (PFS) of 6.4-9.2 months. 15,16As the earliest targeted drugs approved by the United States Food and Drug Administration (FDA), MKIs, including cabozantinib, vandetanib, lenvantinib, and sorafenib, showed comparable clinical responses to chemotherapy but significantly increased toxicity. 17,18In the last decade, immune checkpoint inhibitors (ICIs) have revolutionized NSCLC treatment in patients without oncogenic drivers.However, only a few retrospective studies with small sample sizes have reported the efficacy of ICIs in RET-rearranged NSCLC. 13,19,20First-line application of ICI-based therapies displayed very different results among these studies, with an ORR of 20%-46%, and a PFS of 4.2-11.4months.Recently, the IMpower150 treatment model (bevacizumab+ICIs+chemotherapy) was approved as the standard first-line option for patients with NSCLC without oncogenic drivers or with sensitive EGFR mutations who are resistant to EGFR-TKIs. 21However, the clinical benefits in patients with RET rearrangements are still undetermined.In the era of precision therapy, selective RET-TKIs, including selpercatinib and pralsetinib, significantly improved the prognosis of advanced RET-rearranged NSCLC as demonstrated in the LIBRETTO-001 and ARROW studies. 22,23The first-line ORR and median PFS (mPFS) were 84% and 22 months, respectively, for selpercatinib, and 72% and 13 months, respectively, for pralsetinib.Based on these results, selpercatinib and pralsetinib are the preferred first-line options among the above strategies. 14However, in real-world practice, most patients do not choose RET-TKIs as first-line therapy because of their high cost or specific adverse events such as interstitial pneumonia.For these populations, bevacizumab, immunotherapy, chemotherapy, and their combinations are the standard regimens in the National Comprehensive Cancer Network (NCCN) or The Chinese Society of Clinical Oncology (CSCO) guidelines.However, an optimal scheme beyond RET-TKIs has not yet been defined in the first-line setting.
Here, we aimed to analyze the efficacy and safety of commonly used combination therapies as first-line therapy for RET-rearranged NSCLC.Our study provides realworld evidence for clinical decision-making in patients for whom RET-TKIs cannot be selected.

| Data collection and tumor response assessment
Clinical, pathological, and molecular information of these patients, including age, sex, smoking history, histological types, metastasis sites, RET fusion partners, concomitant gene mutations, programmed death ligand-1 (PD-L1) expression, treatment regimens and duration, and adverse events, were recorded.Tumor response and progression were assessed using the Response Evaluation Criteria in Solid Tumors v1.1, stratified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD).The ORR was defined as the percentage of patients who achieved CR and PR.The sum of the CR, PR, and SD rates was defined as the disease control rate (DCR).PFS was calculated from the start of first-line treatment to the date of disease progression or death from any cause.The final follow-up was conducted on March 25, 2023.Adverse events (AEs) were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0.

| Statistical analysis
SPSS version 25.0 and GraphPad Prism 8 software were used for statistical analyses.Differences in ORR and DCR between the groups were calculated using the chi-square test or Fisher's exact test.The Kaplan-Meier method with the log-rank test was used to summarize and analyze survival rates.Subgroup, univariate, and multivariate survival analyses were performed using the Cox proportional hazards regression model.The Cox proportional hazards regression model was also applied to assess the hazard ratio (HR) and corresponding 95% confidence interval (95% CI).A p < 0.05 was considered statistically significant.A Bonferroni correction was used to compare multiple subgroups (more than two groups).

| Efficacy of different first-line options
First, we analyzed the efficacy of different first-line options, including RET-TKIs, chemotherapy, and chemotherapybased combination therapies.We found that RET-TKIs and combination therapy generated longer PFS, with a mPFS of 16.92 months (95% CI: 5.9-27.9months) and 8.7 months (95% CI: 6.5-11.0months), respectively, compared with chemotherapy alone (mPFS = 5.55 months, 95% CI: 2.4-8.7 months) (Figure 1A).The ORR and DCR in patients receiving RET-TKIs, combination therapy, and chemotherapy were 71.4% and 100%, 17.8% and 94.6%, and 6.7% and 66.7%, respectively (Figure 1B).These results confirmed that RET-TKIs are the preferred option when available.But most patients did not choose RET-TKIs as the first-line therapy due to the high cost.Therefore, chemotherapy-based combination therapy may be an alternative treatment strategy.
Because combination therapy showed beneficial PFS compared with chemotherapy, we further explored which combination scheme was better.Among the 57 patients in the combination therapy group, 15 (26.3%) received I + C, 38 (66.7%) received B + C, and 4 (7.0%) received I + B + C (Figure 2A).PFS was significantly longer in the B + C (p = 0.007) and I + B + C (p = 0.025) groups, but not in the I + C (p = 0.17) group, than in the chemotherapy group (Figure 2B-D).In addition, PFSs in the I + C and B + C groups were comparable (Figure 2E).The mPFS were  12.21, 8.74, and 7.89 months in I + B + C, B + C, and I + C groups, respectively (Figure 2F).These results indicated that the I + B + C treatment model may be a better choice for patients unsuitable for RET-TKIs.The combination of chemotherapy and bevacizumab was superior to ICI treatment.

| Safety
The treatment-related AEs are shown in

| The risk factors for PFS
Univariate and multivariate analyses were performed to identify the risk factors for PFS.In univariate analysis, smoking (p = 0.011), brain metastases (p = 0.009), liver metastases (p = 0.009), and adrenal metastases (p = 0.032) were associated with poor prognosis.In multivariate analysis, smoking (p = 0.031) and brain metastases (p = 0.018) were independent negative indicators of PFS (Table S1).

| DISCUSSION
As a rare subtype of NSCLC, RET rearrangement predicts rapid disease progression and poor outcomes. 24Most patients are diagnosed at advanced stages of the disease, with young age and frequent central nervous system metastasis. 25,26Timely and effective first-line interventions are crucial for improving survival.The development of RET-TKIs has changed the treatment paradigm in this subpolulation.6][27][28] Although RET-TKIs are the preferred firstline options, most patients choose chemotherapy combined T A B L E 2 Adverse events based on different treatment options.with bevacizumab and/or ICIs because of the high cost or specific AEs in real-world data.In this population, no optimal scheme was defined.In this retrospective, multicenter study, we compared the efficacies of the most commonly used first-line treatments, including RET-TKIs, chemotherapy, and chemotherapy-based combination regimens, including I + C, B + C, and I + B + C. Our results suggest that I + B + C might be a preferred option beyond RET-TKIs as a first-line therapy for RET-rearranged NSCLC, followed by a combination of bevacizumab and chemotherapy.This study provides real-world evidence for improved clinical decision-making in first-line settings.Chemotherapy and chemotherapy-based combination therapies, including I + C, B + C, or I + B + C, are commonly selected in clinical practice according to the recommendations of the NCCN or CSCO guidelines. 14,29owever, there is no evidence supporting the choice of these therapies.In the current study, treatment with I + B + C resulted in the highest ORR among these options and significantly improved PFS compared with chemotherapy, suggesting that I + B + C should be given priority when RET-TKIs are unavailable.AEs in the I + B + C group were similar to those in the I + C and B + C groups.Vascular endothelial growth factor suppresses T-cell-mediated immune responses by inhibiting dendritic cell maturation, restricting T-cell infiltration, and inducing suppressive cell components in the tumor microenvironment. 30In preclinical models, bevacizumab reverses the immunosuppressive tumor microenvironment and improves the efficacy of ICI. 31 When used in patients with NSCLC, the combination of bevacizumab with ICIs and chemotherapy significantly prolonged patient survival compared with I + C or B + C in EGFR-mutant but TKI-resistant NSCLC. 32However, to the best of our knowledge, the efficacy of the combined I + B + C treatment in RET-rearranged NSCLC has not yet been determined.Here, we provide the first insights into this field and demonstrate the superior benefits of combining bevacizumab and ICIs.
Immunotherapy has recently revolutionized the NSCLC treatment paradigm.However, its role in RETrearranged NSCLC remains unclear.][35][36] Chemotherapy combined with ICIs has been shown to achieve promising outcomes in upfront treatment of RET-rearranged NSCLC. 37However, real-world data from different groups are controversial.The ORRs ranged between 21% and 46%, and the mPFS ranged from 6.1 to 9.6 months. 13,20In the current study, the combination of ICIs and chemotherapy resulted in numerical, but not statistical, improvements in PFS, suggesting that the scheme should be considered only in certain conditions.Mechanistically, RET rearrangement has been shown to lower tumor mutation burden and PD-L1 expression, 38 explaining their hyporesponsiveness to ICIs.
Surprisingly, patients treated with a combination of ICIs and chemotherapy had a high probability of myocarditis in our cohort.Immunotherapy-related myocarditis occurred in less than 1% of patients in previous studies. 39However, because of the absence of consistent myocardial injury biomarkers and recognizable clinical symptoms, the actual frequency may be underestimated.Immune-related AEs have rarely been reported in RET-rearranged NSCLC.In the RET-MAP study, only one patient (2.4%, 1/41) had pericardial effusion, and no immune-related cardiac toxicity was discovered. 13nother study in which patients were treated with ICIs alone did not report cardiac toxicity. 36Although combined ICIs and chemotherapy increased overall and grade III-V cardiac toxicity in a meta-analysis, 40 the frequency of cardiotoxicity in RET-rearranged NSCLC needs further exploration in a larger patient cohort.In addition, the occurrence of Grade I-II pneumonia in patients receiving Pralsetinib was 21.4% in our study, which is higher than 6% as reported in ARROW study. 22e also observed an earlier onset of pneumonia within 1-3 months after receiving RET-TKI compared with 6 months in the previous study. 41We speculated strict patient screening in clinical trials might decrease the incidence of pneumonia.Also, it may be related to the small number of patient samples.These results suggest that RET-TKI-related pneumonia should be given early attention to avoid serious consequences.
Despite these meaningful findings, our study has some limitations.First, whether the extension of PFS in our study can be converted to a benefit in OS requires further follow-up.Second, the superiority of the I + B + C therapy requires further confirmation with larger sample sizes and prospective studies.Third, due to the insufficient number of cases in the I + C group, we could not identify the subpopulation that could benefit from immunotherapy.

| CONCLUSIONS
In conclusion, I + B + C might be a preferred option beyond RET-TKIs as a first-line therapy for patients with RET-arranged NSCLC.Combination therapy with bevacizumab, rather than ICIs, resulted in favorable survival compared with chemotherapy alone.

AUTHOR CONTRIBUTIONS
Yihui Ge: Data curation (equal); formal analysis (equal); resources (equal); software (equal); writingoriginal draft (equal).Juan Li: Data curation (equal); the combination schemes, B + C (p = 0.007) or I + B + C (p = 0.025) gave beneficial PFS compared with chemotherapy, while I + C treatment (p = 0.169) generated comparable PFS with chemotherapy.In addition, I + B + C treatment had a numerically longer mPFS (12.21 months) compared with B + C (8.74 months) or I + C (7.89 months) schemes.In terms of safety, I + B + C treatment led to the highest frequency of hematological toxicity (50%) and vomiting (75%), but no ≥G3 adverse effect was observed.Conclusions: I + B + C might be a preferred option beyond RET-TKIs in the firstline therapy of RET-arranged NSCLC.Combination with Bevacizumab rather than with ICIs offered favorable survival compared with chemotherapy alone.K E Y W O R D S combination schemes, efficacy, first-line therapy, non-small cell lung cancer, RET rearrangement | 3 of 9 Advanced NSCLC patients harboring RET rearrangements were retrospectively analyzed at six cancer institutions (Shandong Cancer Hospital and Institute, QiLu Hospital of Shandong University, Shandong Provincial Hospital, Yantai Yuhuangding Hospital, Affiliated Hospital of Qingdao University, and Jinan Central Hospital) in China between May 1, 2017, and October 31, 2022.The inclusion criteria were as follows: (1) confirmed NSCLC by cytology or histology; (2) stage IV disease according to the American Joint Committee on Cancer Staging Manual version 8; (3) confirmed RET rearrangement by fluorescence in situ hybridization, next-generation sequencing, or polymerase chain reaction; and (4) patients receiving chemotherapy or chemotherapy-based combination therapy (including ICI combined with chemotherapy [I + C], bevacizumab combined with chemotherapy [B + C], or ICI and bevacizumab combined with chemotherapy [I + B + C]) or RET-TKIs as first-line treatment.Patients with coexisting mutations (e.g., EGFR mutations) were excluded.

F I G U R E 1 F I G U R E 2
Efficacy of first-line therapies.(A) The progression-free survival (PFS) of RET-rearranged NSCLC patients based on different first-line therapies.The mPFS in patients receiving RET-TKIs, chemotherapy-based combination therapy, and chemotherapy alone were 16.92, 8.7, and 5.55 months, respectively.(B) The ORR and DCR of patients treated with different first-line therapies.The ORRs were 71.4%, 17.8%, and 6.7% in patients receiving RET-TKIs, combination therapy, and chemotherapy, respectively.RET-TKIs, selective RET-tyrosine kinase inhibitor; ORR, objective response rate; DCR, disease control rate.Efficacy of different combination therapies.(A) The frequency of patients receiving different combination therapies.2/3 of patients received B + C and only 7% of patients received I + B + C treatment.(B) Patients treated with B + C had markedly longer progression-free survival (PFS) compared with those with chemotherapy alone (p = 0.007).(C) Compared with chemotherapy, I + B + C treatment generated a longer PFS (p = 0.025).(D) I + C treatment generated numerical but not statistical improvement of PFS compared with chemotherapy alone (p = 0.17).(E) Treatment with I + C and B + C displayed comparable PFS (p = 0.377).(F) The mPFS (medium progression-free survival) in I+B+C, B+C, and I+C group were 12.21, 8.74, and 7.89 months, respectively.
T A B L E 1Abbreviation: n, number.

Table 2 .
The most common grade I/II AE in the RET-TKI group was pneumonia (n = 3; 21.4%).Vomiting and hematologic toxicity were the most common grade I/II AEs in the chemotherapy and combination therapy groups.The I + B + C group had the highest incidence of vomiting, followed by the I + C and B + C groups.Hematologic toxicity normally appeared in the B + C group instead of the I + B + C and I + C groups, and the most common grade III/IV adverse events in chemotherapy and combination therapy were hematologic toxicity.Compared with the I + B + C and B + C groups, the I + C group had the highest incidence.Myocarditis occurred in two patients (13.3%) in the I + C group, resulting in discontinued ICI usage.AEs leading to treatment discontinuation did not occur in the I + B + C and B + C groups.